TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc
TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc
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DOI：
10.1074/jbc.m601969200
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发表时间：
2006-09-01
影响因子：
4.8
通讯作者：
Risbud, Makarand V.
中科院分区：
文献类型：
--
作者：
Tsai, Tsung-Ting;Danielson, Keith G.;Risbud, Makarand V.
髓核是富含聚集蛋白聚糖的水合组织，其允许椎间盘抵抗压缩负荷。通过聚集蛋白聚糖分子的许多带电荷的糖氨基聚糖侧链介导的椎间盘渗透压升高来适应负荷。本研究的目的是确定在髓核细胞中的神经调节蛋白TonEBP的功能作用。我们发现TonEBP及其下游靶基因在椎间盘组织中稳健表达。高于330 mosmol/kg，培养的髓核细胞上调靶基因TauT、BGT-1和SMIT;高于450 mosmol/kg，HSP-70的表达增加。在高渗介质中，TauT和热休克蛋白-70（HSP-70）报告活性被激活，TonEBP与TonE基序的结合增加。当细胞转染显性阴性形式的TonEBP（DN-TonEBP）有TauT和HSP-70报告基因表达的抑制; pTonEBP增强报告基因的表达。此外，在高渗介质中，DN-TonEBP的强制表达诱导细胞凋亡。我们使用小干扰RNA技术抑制TonEBP，并注意到在等渗和高渗介质中TauT报告活性降低。最后，我们报告聚集蛋白聚糖启动子包含两个保守的TonE基序。为了评估这些基序的重要性，我们过表达DN-TonEBP并使用小干扰RNA部分沉默TonEBP。这两种方法导致聚集蛋白聚糖启动子活性的抑制。可以得出结论，TonEBP允许椎间盘细胞适应高渗环境，同时自动调节产生独特的细胞外环境的分子的表达。
The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmoregulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment.